AU2007280846B2 - Branched hyaluronic acid and method of manufacture - Google Patents
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- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q7/00—Preparations for affecting hair growth
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Abstract
A branched hyaluronic acid, wherein the linear backbone comprises hyaluronic acid in which one or more -Acetyl-Glucosamine has been deacetylated to Glucosamine, with branching sidechain(s) covalently linked to the primary amine(s) of said deacetylated Glucosamine thus forming a secondary amine(s); a precursor for producing said branched hyaluronic acid; and a method for producing said branching hyaluronic acid
Description
TITLE: BRANCHED HYALURONIC ACID AND METHOD OF MANUFACTURE FIELD OF THE INVENTION The present invention relates to the branching of deacetylated hyaluronic acid 5 (deHA) by reductive alkylation, to the branched hyaluronic acid as such, and to its applications and uses, particularly in the cosmetics and biomedical industries. BACKGROUND OF THE INVENTION Any discussion of the prior art throughout the specification should in no way be 10 considered as an admission that such prior art is widely known or forms part of common general knowledge in the field. The most abundant heteropolysaccharides of the body are the glycosaminoglycans. Glycosaminoglycans are unbranched carbohydrate polymers, consisting of repeating disaccharide units (only keratan sulphate is branched in the core 15 region of the carbohydrate). The disaccharide units generally comprise, as a first saccharine unit, one of two modified sugars - N-acetylgalactosamine (GaINAc) or N acetylglucosamine (GIcNAc). The second unit is usually an uronic acid, such as glucuronic acid (GlcUA) or iduronate. Glycosaminoglycans are negatively charged molecules, and have an extended 20 conformation that imparts high viscosity when in solution. Glycosaminoglycans are located primarily on the surface of cells or in the extracellular matrix. Glycosaminoglycans also have low compressibility in solution and, as a result, are ideal as a physiological lubricating fluid, e.g., joints. The rigidity of glycosaminoglycans provides structural integrity to cells and provides passageways between cells, allowing 25 for cell migration. The glycosaminoglycans of highest physiological importance are hyaluronan, chondroitin sulfate, heparin, heparan sulfate, dermatan sulfate, and keratan sulfate. Most glycosaminoglycans bind covalently to a proteoglycan core protein through specific oligosaccharide structures. Hyaluronan forms large aggregates with certain proteoglycans, but is an exception as free carbohydrate chains form non 30 covalent complexes with proteoglycans. Numerous roles of hyaluronan in the body have been identified (see, Laurent T. C. and Fraser J. R. E., 1992, FASEB J. 6: 2397-2404; and Toole B.P., 1991, "Proteoglycans and hyaluronan in morphogenesis and differentiation." In: Cell Biology 1 of the Extracellular Matrix, pp. 305-341, Hay E. D., ed., Plenum, New York). Hyaluronan is present in hyaline cartilage, synovial joint fluid, and skin tissue, both dermis and epidermis. Hyaluronan is also suspected of having a role in numerous physiological functions, such as adhesion, development, cell motility, cancer, 5 angiogenesis, and wound healing. Due to the unique physical and biological properties of hyaluronan, it is employed in eye and joint surgery and is being evaluated in other medical procedures. The terms "hyaluronan" or "hyaluronic acid" are used in literature to mean acidic polysaccharides with different molecular weights constituted by residues of D-glucuronic 10 and N-acetyl-D-glucosamine acids, which occur naturally in cell surfaces, in the basic 1 a WO 2008/014787 PCT/DK2007/000358 extracellular substances of the connective tissue of vertebrates, in the synovial fluid of the joints, in the endobulbar fluid of the eye, in human umbilical cord tissue and in cocks' combs. The term "hyaluronic acid" is in fact usually used as meaning a whole series of polysaccharides with alternating residues of D-glucuronic and N-acetyl-D-glucosamine acids 5 with varying molecular weights or even the degraded fractions of the same, and it would therefore seem more correct to use the plural term of "hyaluronic acids". The singular term will, however, be used all the same in this description; in addition, the abbreviation "HA" will frequently be used in place of this collective term. HA plays an important role in the biological organism, as a mechanical support for the 10 cells of many tissues, such as the skin, tendons, muscles and cartilage, it is a main component of the intercellular matrix. HA also plays other important parts in the biological processes, such as the moistening of tissues, and lubrication. HA may be extracted from the above mentioned natural tissues, although today it is preferred to prepare it by microbiological methods to minimize the potential risk of 15 transferring infectious agents, and to increase product uniformity, quality and availability. HA and its various molecular size fractions and the respective salts thereof have been used as medicaments, especially in treatment of arthropathies, as an auxiliary and/or substitute agent for natural organs and tissues, especially in ophtalmology and cosmetic surgery, and as agents in cosmetic preparations. Products of hyaluronan have also been 20 developed for use in orthopaedics, rheumatology, and dermatology. HA may also be used as an additive for various polymeric materials used for sanitary and surgical articles, such as polyurethanes, polyesters etc. with the effect of rendering these materials biocompatible. De-N-acetylation of HA with hydrazine has been described in the literature (Crescenzi 25 et al. (2002) New cross-linked and sulfated derivatives of partially deacetylated hyaluronan: Synthesis and preliminary characterization, Biopolymers 64, 86-94). Branching of chitosans by reductive HONO degradation and reductive N-alkylation has been described (Tommeraas et al. (2002) Carbohydrate Research 337, 2455-2462). 30 SUMMARY OF THE INVENTION There is a need to develop novel derivatives of biologically compatible biopolymers with desirable properties, for instance, improved visco-elastic properties compared to linear HA, e.g., less sensible to shear thinning or changes in ionic strength, or lower viscosity than linear HA of same MW. These properties are believed to be of value in biomechanical 35 implants and in advanced cosmetic, biomedical and pharmaceutical formulations. Other properties of interest are the improved ability to stabilize foam and the ability to blend with non-hydrophilic materials, such as is used typically in cosmetics products. 2 In a first aspect, the present invention provides a branched hyaluronic acid, wherein the linear backbone comprises hyaluronic acid in which one or more N-Acetyl Glucosamine has been deacetylated to Glucosamine, with branching sidechain(s) covalently linked to the primary amine(s) of said deacetylated Glucosamine thus 5 forming a secondary amine(s). In some preferred embodiments the invention relates to a precursor or intermediate molecule that enters into the method of the invention, namely a partially or fully deacetylated hyaluronic acid (dHA), wherein one or more N-Acetyl-Glucosamine has been deacetylated to Glucosamine. 10 Accordingly, in a second aspect, the invention relates to a method for producing a branced hyaluronic acid, the method comprising the steps of: a) providing a linear hyaluronic acid backbone, wherein one or more N-Acetyl Glucosamine has been deacetylated to Glucosamine; and b) reacting a biocompatible polymer comprising at least one free reducing aldehyde 15 group with the primary amine(s) of the one or more Glucosamine of (a) by reductive N-alkylation; to form a branched hyaluronic acid. In some preferred embodiments the invention provides a method for producing a branched hyaluronic acid, the method comprising the steps of: a) providing a linear hyaluronic acid backbone, wherein one or more N-Acetyl 20 Glucosamine has been deacetylated to Glucosamine; and b) reacting a biocompatible polymer comprising at least one free reducing aldehyde group with the primary amine(s) of the one or more Glucosamine of (a) by reductive N-alkylation; to form a branched hyaluronic acid. In a third aspect, the invention relates to a composition comprising a branched 25 hyaluronic acid as defined in the first aspect, and an active ingredient, preferably the active ingredient is a pharmacologically active agent. A fourth aspect of the invention relates to a pharmaceutical composition comprising an effective amount of a branched hyaluronic acid as defined in the first aspect, together with a pharmaceutically acceptable carrier, excipient or diluent. 30 A fifth aspect relates to a pharmaceutical composition comprising an effective amount of a branched hyaluronic acid as defined in the first aspect as a vehicle, together with a pharmacologically active agent. 3 In some preferred embodiments the invention relates to a cosmetic article comprising as an active ingredient an effective amount of a branched hyaluronic acid as defined in the first aspect or a composition as defined in any one of the third, fourth or fifth aspects. 5 In a sixth aspect, the invention relates to a sanitary, medical or surgical article comprising a branched hyaluronic acid as defined in the first aspect, or a composition as defined in any one of the third, fourth or fifth aspects, preferably the article is a diaper, a sanitary towel, a surgical sponge, a wound healing sponge, or a part comprised in a band aid or other wound dressing material. 10 In a seventh aspect, the present invention relates to a medicament capsule or microcapsule comprising a branched hyaluronic acid as defined in the first aspect or a composition as defined in any one of the third, fourth or fifth aspects. In an eighth aspect, the present invention relates to use of a branched hyaluronic acid as defined in the first aspect or a composition as defined in any one of 15 the third, fourth or fifth aspects for the manufacture of a medicament for the treatment of osteoarthritis; for an ophthalmological treatment; for the treatment of cancer; for the treatment of a wound; or for angiogenesis. In a ninth aspect, the present invention provides a method for the treatment of osteoarthritis; for an ophthalmological treatment; for the treatment of cancer; for the 20 treatment of a wound; or for angiogenesis comprising administering a branched hyaluronic acid as defined in the first aspect or a composition as defined in any one of the third, fourth or fifth aspects to a subject in need thereof Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an 25 inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". An important aspect relates to a medicament capsule or microcapsule comprising a branched hyaluronic acid as defined in the first aspect or a composition as defined in any of the third to fifth aspects. 30 Final aspects of the invention relate to methods of performing procedures in ophtalmology, in the treatment of osteoarthritis or cancer, of treating a wound, of performing 3a WO 2008/014787 PCT/DK2007/000358 dermal or transdermal administration of a pharmacologically active agent, or dermal administration of a cosmetic, the improvement which comprises the use of a branched hyaluronic acid as defined in the first aspect, or a composition as defined in any of the third to sixth aspects. 5 A number of aspects relate to uses of a branched hyaluronic acid as defined in any of the first aspects or a composition as defined in any of the fourth to sixth aspects for the manufacture of a medicament for the treatment of osteoarthritis, cancer, the manufacture of a medicament for an ophtalmological treatment, the manufacture of a medicament for the treatment of a wound, the manufacture of a medicament for angiogenesis, or the 10 manufacture of a moisturizer. BRIEF DESCRIPTION OF DRAWINGS Figure 1 shows a plot of the contraction factor 'g' as function of molecular weight of the hyaluronic acid (g = (Rg,branched) 2 /(Rg inear) 2 ). A g-value below 1 indicates branching of the 15 polymer. As can be seen in the figure, the contraction factor increases with increasing molecular weight, thus confirming the preparation of branched HA. Samples 2.a, 2.b and 2.c correspond to samples A, B and C of Example 2 after the branching reaction has been carried out. 20 DETAILED DESCRIPTION OF THE INVENTION Hyaluronic Acid "Hyaluronic acid" is defined herein as an unsulphated glycosaminoglycan composed of repeating disaccharide units of N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcUA) linked together by alternating beta-1,4 and beta-1,3 glycosidic bonds. Hyaluronic 25 acid is also known as hyaluronan, hyaluronate, or HA. The terms hyaluronan and hyaluronic acid are used interchangeably herein to describe the molecule as such, as well as any kind of salt thereof. Rooster combs are a significant commercial source for hyaluronan. Microorganisms are an alternative source. U.S. Patent No. 4,801,539 discloses a fermentation method for 30 preparing hyaluronic acid involving a strain of Streptococcus zooepidemicus with reported yields of about 3.6 g of hyaluronic acid per liter. European Patent No. EP0694616 discloses fermentation processes using an improved strain of Streptococcus zooepidemicus with reported yields of about 3.5 g of hyaluronic acid per liter. As disclosed in WO 03/054163 (Novozymes), which is incorporated herein in its entirety, hyaluronic acid or salts thereof may 35 be recombinantly produced, e.g., in a Gram-positive Bacillus host. Hyaluronan synthases have been described from vertebrates, bacterial pathogens, and algal viruses (DeAngelis, P. L., 1999, Cell. Mol. Life Sci. 56: 670-682). WO 99/23227 4 WO 2008/014787 PCT/DK2007/000358 discloses a Group I hyaluronate synthase from Streptococcus equisimiis. WO 99/51265 and WO 00/27437 describe a Group II hyaluronate synthase from Pasturella multocida. Ferretti et al. disclose the hyaluronan synthase operon of Streptococcus pyogenes, which is composed of three genes, hasA, hasB, and hasC, that encode hyaluronate synthase, UDP 5 glucose dehydrogenase, and UDP-glucose pyrophosphorylase, respectively (Proc. Nat/. Acad. Sci. USA. 98, 4658-4663, 2001). WO 99/51265 describes a nucleic acid segment having a coding region for a Streptococcus equisimilis hyaluronan synthase. Since the hyaluronan of a recombinant Bacillus cell is expressed directly to the culture medium, a simple process may be used to isolate the hyaluronan from the culture 10 medium. First, the Bacillus cells and cellular debris are physically removed from the culture medium. The culture medium may be diluted first, if desired, to reduce the viscosity of the medium. Many methods are known to those skilled in the art for removing cells from culture medium, such as centrifugation or microfiltration. If desired, the remaining supernatant may then be filtered, such as by ultrafiltration, to concentrate and remove small molecule 15 contaminants from the hyaluronan. Following removal of the cells and cellular debris, a simple precipitation of the hyaluronan from the medium is performed by known mechanisms. Salt, alcohol, or combinations of salt and alcohol may be used to precipitate the hyaluronan from the filtrate. Once reduced to a precipitate, the hyaluronan can be easily isolated from the solution by physical means. The hyaluronan may be dried or concentrated from the 20 filtrate solution by using evaporative techniques known to the art, such as spray drying. The first aspect of the invention relates to a branched hyaluronic acid, wherein the linear backbone comprises hyaluronic acid in which one or more N-Acetyl-Glucosamine has been deacetylated to Glucosamine, with branching sidechain(s) covalently linked to the 25 primary amine(s) of said deacetylated Glucosamine thus forming a secondary amine(s). In a preferred embodiment the branching sidechain(s) comprise a biocompatible polymer which preferably comprises hyaluronic acid. The second aspect of the invention relates to an intermediary or precursor molecule necessary for the manufacture of the branched HA of the first aspect in the method of the 30 third aspect, which is a partially or fully deacetylated hyaluronic acid (dHA), wherein one or more N-Acetyl-Glucosamine has been deacetylated to Glucosamine. Preferably the one or more N-Acetyl-Glucosamine has been deacetylated by chemical and/or enzymatic treatment, for instance by using hydrazine monohydrate together with hydrazine sulphate, or by using an enzyme having HA deacetylase activity. 35 A preferred embodiment relates to the dHA of the second aspect, wherein 50% or less of the N-Acetyl-Glucosamines in the linear hyaluronic acid backbone have been deacetylated to Glucosamine, preferably 40% or less, more preferably 30% or less, still more 5 WO 2008/014787 PCT/DK2007/000358 preferably 20% or less, 10% or less, or most preferably 5% or less of the N-Acetyl Glucosamines in the linear backbone have been deacetylated to Glucosamine. Another preferred embodiment relates to the dHA of the second aspect, which has an average molecular weight in the range of 10 -.3,000 kiloDalton, preferably 20 - 2,000 kDa, 5 and most preferably 20 - 1,000 kDa, or even smaller, such as 20 - 900 kDa, 20 - 800 kDa, 20 - 700 kDa, 20 - 600 kDa, 20 - 500 kDa, 20 - 400 kDa, 20 - 300 kDa, 20 - 200 kDa, or 20-100kDa. Molecular weight The level of hyaluronic acid may be determined according to the modified carbazole 10 method (Bitter and Muir, 1962, Anal Biochem. 4: 330-334). Moreover, the average molecular weight of the hyaluronic acid may be determined using standard methods in the art, such as those described by Ueno et al., 1988, Chem. Pharm. Bull. 36, 4971-4975; Wyatt, 1993, Anal. Chim. Acta 272: 1-40; and Wyatt Technologies, 1999, "Light Scattering University DAWN Course Manual" and "DAWN EOS Manual" Wyatt Technology Corporation, Santa Barbara, 15 California. It may be advantageous in some instances to first reduce the average molecular weight of the linear hyaluronic acid backbone or salts thereof. For instance, it has been stated by several manufacturers of so-called low-molecular weight fractions of hyaluronic acid, that it is capable of penetrating the skin barrier to reestablish the natural content of 20 hyaluronic acid in the skin, therefore such fractions are particularly suitable for cosmetic compositions sold as anti-skin-ageing and anti-wrinkle agents. For food applications, low MW hyaluronic acid has been shown to penetrate the gastrointestinal barrier, thereby increasing its bioavailability. Finally, low MW hyaluronic acid exhibits anti-inflammatory effect and has potential applications in the treatment of inflammatory diseases. A reduction of the average 25 molecular weight of a hyaluronic acid or derivative or salt thereof may be achieved by standard methods in the art, such as, simple heat treatment, enzymatic degradation, ultrasound sonication, or acid hydrolysis. See, e.g., US patent 6,020,484, which describes an ultrasonication technique of HA including NaOCI as additive, and T. Miyazaki et al. (2001) Polymer Degradation and Stability, 74: 77-85. 30 The third aspect of the invention relates to a method for producing a branched hyaluronic acid, the method comprising the steps of: a) providing a linear hyaluronic acid backbone, wherein one or more N-Acetyl-Glucosamine has been deacetylated to Glucosamine; and 35 b) reacting a biocompatible polymer comprising at least one free reducing aldehyde group with the primary amine(s) of the one or more Glucosamine of (a) by reductive N alkylation; to form a branched hyaluronic acid. 6 WO 2008/014787 PCT/DK2007/000358 Preferably in the method of the third aspect 50% or less of the N-Acetyl Glucosamines in the linear hyaluronic acid backbone have been deacetylated to Glucosamine, preferably 40% or less, more preferably 30% or less, still more preferably 20% or less, 10% or less, or most preferably 5% or less of the N-Acetyl-Glucosamines in the 5 linear backbone have been deacetylated to Glucosamine. In a preferred embodiment of the third aspect the linear hyaluronic acid backbone has an average molecular weight in the range of 10 - 3,000 kiloDalton, preferably 20 - 2,000 kDa, and most preferably 20 - 1,000 kDa, or even smaller, such as 20 - 900 kDa, 20 - 800 kDa, 20 - 700 kDa, 20 - 600 kDa, 20 - 500 kDa, 20 - 400 kDa, 20 - 300 kDa, 20 - 200 kDa, or 10 20 - 100 kDa. Biocompatible polymers are well-known in the art, and encompasses all kinds of polymers, both naturally produced or synthetically manufactured, which are capable of being degraded and metabolized by an organism, such as a human, without any toxic or unhealthy effects. 15 Another preferred embodiment relates to the method of the third aspect, wherein the reductive N-alkylation reaction is done in the presence of Sodium Cyanoborohydride, NaCNBH 3 , preferably at a pH value in the range of 4 - 10, preferably 5 - 9, more preferably 6 - 8, and most preferably at approximately 7. 20 Other ingredients In a preferred embodiment, the compositions comprising a branched HA of the invention may also comprise other ingredients, preferably one or more active ingredient, preferably one or more pharmacologically active substance, and also preferably a water soluble excipient, such as lactose. 25 Non-limiting examples of an active ingredient or pharmacologically active substance which may be used in the present invention include protein and/or peptide drugs, such as, human growth hormone, bovine growth hormone, porcine growth hormone, growth homorne releasing hormone/peptide, granulocyte-colony stimulating factor, granulocyte macrophage colony stimulating factor, macrophage-colony stimulating factor, erythropoietin, bone 30 morphogenic protein, interferon or derivative thereof, insulin or derivative thereof, atriopeptin III, monoclonal antibody, tumor necrosis factor, macrophage activating factor, interleukin, tumor degenerating factor, insulin-like growth factor, epidermal growth factor, tissue plasminogen activator, factor IIV, factor lIlV, and urokinase. A water-soluble excipient may be included for the purpose of stabilizing the active 35 ingredient(s), such excipient may include a protein, e.g., albumin or gelatin; an amino acid, such as glycine, alanine, glutamic acid, arginine, lysine and a salt thereof; carbohydrate such as glucose, lactose, xylose, galactose, fructose, maltose, saccharose, dextran, mannitol, 7 WO 2008/014787 PCT/DK2007/000358 sorbitol, trehalose and chondroitin sulphate; an inorganic salt such as phosphate; a surfactant such as TWEEN@ (ICI), poly ethylene glycol, and a mixture thereof. The excipient or stabilizer may be used in an amount ranging from 0.001 to 99% by weight of the product. Several aspects of the invention relate to various compositions and pharmaceuticals 5 comprising, among other constituents, an effective amount of the product as defined in the first aspect, and an active ingredient, preferably the active ingredient is a pharmacologically active agent; a pharmaceutically acceptable carrier, excipient or diluent, preferably a water soluble excipient, and most preferably lactose. In addition, aspects of the invention relate to articles comprising a branched HA as 10 defined in the first aspect or a composition as defined in the aspects and embodiments above, e.g., a cosmetic article, a sanitary article, a medical or surgical article. In a final aspect the invention relates to a medicament capsule or microcapsule comprising a product as defined in the first aspect or a composition as defined in other aspects and embodiments of the invention. 15 EXAMPLES Example I - Preparation of deacetylated HA HA (6.0 g) was dissolved in hydrazine monohydrate (300 mL) together with hydrazine sulphate (3.0 g) and left on stirring for 92 hours at 55 0 C. The resulting product was recovered 20 by precipitation with cold ethanol (350 mL). Saturated aqueous NaCl (5 mL) was added to improve precipitation. The recovered precipitate was washed in fresh ethanol (250 mL) and recovered by centrifugation (3000g, 10 minutes). The recovered material (780 mg) was found to be deacetylated HA (degree of deacetylation 13%). Example 2 - Preparation of degraded deacetylated HA 25 Deacetylated HA (deHA) prepared as described in example 1 was degraded by nitrous acid using the following procedures: Three samples A, B and C of deHA (100 mg) were dissolved in 1% aqueous acetic acid (6 mL). Nitrous acid (NaNO 2 ) was added according to Table 1. The solutions were left for 4 hours in darkness before pH was adjusted to approximately 7. 30 Table 1. Amounts of nitrous acid added to samples A, B and C and the resulting number of covalent bonds broken. 8 WO 2008/014787 PCT/DK2007/000358 Sample Amount NaNO 2 % bonds broken (mg/100 mg HA) A 1.38 4 B 0.690 2 C 0.345 0.5 Example 3 - Preparation of branched HA The samples A, B and C of degraded deHA prepared in example 2 were branched by reductive alkylation using the following procedure: Sodium Cyanoborohydride, NaCNBH 3 5 (20.41 mg) were added to the pH-adjusted solutions from Example 2. The reactions were left to proceed for 48 hours (stirring) before stopped, and products where recovered by dialysis against deionized water (MWCO 12-14 kDa) followed by freeze drying. Example 4 - Analysis by SEC-MALLS-visc The branched product-samples 2.a, 2.b, and 2.c prepared in Example 3 10 (corresponding to samples A, B and C of Example 2) were analysed by size exclusion chromatography with the following on-line detectors: MALLS (multi-angle laser light scattering), RI (refractive index) and visc (intrinsic viscosity detector). This was used to evaluate the conformation and molecular weight of the produced materials, i.e., to see if they were branched. Figure 1 shows a plot of the contraction factor g as function of molecular 15 weight (g = (Rg,branched) 2 /(Rg inear)2). A g-value below 1 indicates branching of the polymer. As can be seen, the contraction factor increases with increasing molecular weight, proving the preparation of branched HA. Table 2 summarizes the properties of the branched HA samples 2.a, 2.b and 2.c. The parameter 'a' in table 2, is obtained from a plot of log Rg vs. log M,. This parameter gives 20 information about the conformation of the polymer in the solvent used (in general; random coil: 0.5-0.6, stiff rod: 1.0 and sphere: 0.33). For the starting material (regular HA), this value is 0.5-0.6, as expected for a random coil conformation. A value around 0.25 is expected for a randomly hyper-branched polymer, giving a further indication that samples 2.a, 2.b and 2.c are branched products. 25 Table 2. Main characteristics of branched HA samples 2.a, 2.b and 2.c (M" is the weight average molecular weight, PDI, the polydispersity index, Rg, the z- average radius of gyration, [q] the weight-average intrinsic viscosity and a, the exponent of the equation Rg ~Ma) 30 9 WO 2008/014787 PCT/DK2007/000358 Samples M. PDI Rg [q] a (kDa) (M./M.) (nm) (dUg) (Rg ~Ma) 2.a 131 1.89 39.3 511 0.24 2.b 199 2.04 57.9 772 0.21 2.c 273 2.15 55.7 492 0.30 10
Claims (20)
1. A branched hyaluronic acid, wherein the linear backbone comprises hyaluronic acid in which one or more N-Acetyl-Glucosamine has been deacetylated to 5 Glucosamine, with branching sidechain(s) covalently linked to the primary amine(s) of said deacetylated Glucosamine thus forming a secondary amine(s).
2. The branched hyaluronic acid of claim 1, wherein the branching sidechain(s) comprise a biocompatible polymer. 10
3. The branched hyaluronic acid of claim 2, wherein the biocompatible polymer comprises hyaluronic acid.
4. The branched hyaluronic acid of claim 1, wherein 50% or less of the N-Acetyl 15 Glucosamines in the linear hyaluronic acid backbone have been deacetylated to Glucosamine, preferably 40% or less, more preferably 30% or less, still more preferably 20% or less, or 10% or less.
5. The branched hyaluronic acid of claim 4, wherein 5% or less of the N-Acetyl 20 Glucosamines in the linear backbone have been deacetylated to Glucosamine.
6. A method for producing a branched hyaluronic acid, the method comprising the steps of: a) providing a linear hyaluronic acid backbone, wherein one or more N-Acetyl 25 Glucosamine has been deacetylated to Glucosamine; and b) reacting a biocompatible polymer comprising at least one free reducing aldehyde group with the primary amine(s) of the one or more Glucosamine of (a) by reductive N-alkylation; to form a branched hyaluronic acid. 30
7. The method of claim 6, wherein 50% or less of the N-Acetyl-Glucosamines in the linear hyaluronic acid backbone have been deacetylated to Glucosamine, preferably 40% or less, more preferably 30% or less, still more preferably 20% or less, 10% or less. 35
8. The method of claim 7, wherein 5% or less of the N-Acetyl-Glucosamines in the linear backbone have been deacetylated to Glucosamine. - 12
9. The method of any one of claims 6 to 8, wherein the linear hyaluronic acid backbone has an average molecular weight in the range of 10 - 3,000 kiloDalton. 5
10. The method of any one of claims 6 to 8, wherein the linear hyaluronic acid backbone has an average molecular weight in the range of 20 - 2,000 kDa.
11. The method of any one of claims 6 to 8, wherein the linear hyaluronic acid backbone has an average molecular weight in the range of 20 - 1,000 kDa. 10
12. The method of any one of claims 6 to 7, wherein the biocompatible polymer comprises hyaluronic acid.
13. A composition comprising a branched hyaluronic acid as defined in any one of 15 claims 1 to 5 and an active ingredient, preferably the active ingredient is a pharmacologically active agent.
14. A pharmaceutical composition comprising an effective amount of a branched hyaluronic acid as defined in any one of claims 1 to 5, together with a pharmaceutically 20 acceptable carrier, excipient or diluent.
15. A pharmaceutical composition comprising an effective amount of a branched hyaluronic acid as defined in any one of claims 1 to 5 as a vehicle, together with a pharmacologically active agent. 25
16. A sanitary, medical or surgical article comprising a branched hyaluronic acid as defined in any one of claims 1 to 5 or a composition as defined in any one of claims 13 to 15; preferably the article is a diaper, a sanitary towel, a surgical sponge, a wound healing sponge, or a part comprised in a band aid or other wound dressing material. 30
17. A medicament capsule or microcapsule comprising a branched hyaluronic acid as defined in any one of claims 1 to 5 or a composition as defined in any one of claims 13 to 15. 35
18. Use of a branched hyaluronic acid as defined in any one of claims 1 to 5 or a composition as defined in any one of claims 13 to 15 for the manufacture of a - 13 medicament for the treatment of osteoarthritis; for an ophthalmological treatment; for the treatment of cancer; for the treatment of a wound; or for angiogenesis.
19. A method for the treatment of osteoarthritis; for an ophthalmological treatment; 5 for the treatment of cancer; for the treatment of a wound; or for angiogenesis comprising administering a branched hyaluronic acid as defined in any one of claims 1 to 5 or a composition as defined in any one of claims 13 to 15 to a subject in need thereof. 10
20. A branched hyaluronic acid; a method for producing a branched hyaluronic acid; a composition comprising a branched hyaluronic acid; a pharmaceutical composition comprising an effective amount of a branched hyaluronic acid; a sanitary, medical or surgical article comprising a branched hyaluronic acid; a medicament capsule or microcapsule comprising a branched hyaluronic acid substantially as herein described 15 with reference to any one or more of the examples but excluding comparative examples.
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TWI561535B (en) | 2011-10-06 | 2016-12-11 | Bvw Holding Ag | Copolymers of hydrophobic and hydrophilic segments that reduce protein adsorption |
CZ2012842A3 (en) | 2012-11-27 | 2014-08-20 | Contipro Biotech S.R.O. | C6-C18-acylated hyaluronate-based nanomicellar composition, process for preparing C6-C18-acylated hyaluronate, process for preparing nanomicellar composition and stabilized nanomicellar composition as well as use thereof |
CZ2014150A3 (en) | 2014-03-11 | 2015-05-20 | Contipro Biotech S.R.O. | Conjugates of hyaluronic acid oligomer or salts thereof, process of their preparation and use |
US10363595B2 (en) | 2014-06-09 | 2019-07-30 | Hyperion Materials & Technologies (Sweden) Ab | Cemented carbide necking tool |
CZ2014451A3 (en) | 2014-06-30 | 2016-01-13 | Contipro Pharma A.S. | Antitumor composition based on hyaluronic acid and inorganic nanoparticles, process of its preparation and use |
CZ309295B6 (en) | 2015-03-09 | 2022-08-10 | Contipro A.S. | Self-supporting, biodegradable film based on hydrophobized hyaluronic acid, method of its preparation and use |
CZ306479B6 (en) | 2015-06-15 | 2017-02-08 | Contipro A.S. | A method of crosslinking polysaccharides by using photolabile protecting groups |
CZ306662B6 (en) | 2015-06-26 | 2017-04-26 | Contipro A.S. | Sulphated polysaccharides derivatives, the method of their preparation, the method of their modification and the use |
CZ308106B6 (en) | 2016-06-27 | 2020-01-08 | Contipro A.S. | Unsaturated derivatives of polysaccharides, preparing and using them |
KR101969446B1 (en) * | 2017-06-30 | 2019-04-16 | 한양대학교 산학협력단 | Hyaluronate-based materials with high water retention properties |
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JPS60251898A (en) | 1984-05-25 | 1985-12-12 | Shiseido Co Ltd | Preparation of hyaluronic acid by fermentation method |
US5510418A (en) * | 1988-11-21 | 1996-04-23 | Collagen Corporation | Glycosaminoglycan-synthetic polymer conjugates |
JP2739232B2 (en) * | 1989-04-14 | 1998-04-15 | チッソ株式会社 | Method of using cellulose gel having biological affinity |
IT1271001B (en) | 1994-07-26 | 1997-05-26 | Poli Ind Chimica Spa | HYALURONIC ACID PREPARATION PROCEDURE BY STREPTOCOCCUS FERMENTATION |
IT1282219B1 (en) | 1995-12-20 | 1998-03-16 | Fidia Advanced Biopolymers Srl | COMBINED CHEMICAL-PHYSICAL PROCESS FOR THE PREPARATION OF LOW MOLECULAR WEIGHT HYALURONIC ACID FRACTIONS CHARACTERIZED BY LOW |
CN1322121C (en) | 1997-10-31 | 2007-06-20 | 俄克拉何马大学董事会 | Hyaluronan synthase gene and uses thereof |
CN101275143A (en) | 1998-04-02 | 2008-10-01 | 俄克拉何马大学董事会 | Nucleic acid encoding hyaluronan synthase and methods of use |
IT1303735B1 (en) * | 1998-11-11 | 2001-02-23 | Falorni Italia Farmaceutici S | CROSS-LINKED HYALURONIC ACIDS AND THEIR MEDICAL USES. |
EP1129209B1 (en) | 1998-11-11 | 2009-01-21 | The Board Of Regents Of The University Of Oklahoma | Polymer grafting by polysaccharide synthases |
GB9902652D0 (en) * | 1999-02-05 | 1999-03-31 | Fermentech Med Ltd | Process |
JP2000248002A (en) * | 1999-02-19 | 2000-09-12 | Denki Kagaku Kogyo Kk | Self-crosslinked hyaluronic acid, its production and its use |
GB2358637A (en) * | 2000-01-27 | 2001-08-01 | Btg Int Ltd | Chitosan condensation products with a bisulphite addition compound |
IT1317358B1 (en) * | 2000-08-31 | 2003-06-16 | Fidia Advanced Biopolymers Srl | CROSS-LINKATED DERIVATIVES OF HYALURONIC ACID. |
KR100375299B1 (en) * | 2000-10-10 | 2003-03-10 | 주식회사 엘지생명과학 | Crosslinked derivatives of hyaluronic acid by amide formation and their preparation methods |
ITTS20010013A1 (en) * | 2001-06-04 | 2002-12-04 | Ct Ricerche Poly Tec H A R L S | NEW HALURONAN DERIVATIVES. |
DK1572895T3 (en) | 2001-12-21 | 2011-07-11 | Novozymes Biopharma Dk As | Methods for preparing hyaluronan in a recombinant host cell |
TW200307011A (en) * | 2002-04-18 | 2003-12-01 | Chugai Pharmaceutical Co Ltd | Hyaluronic acid modifier |
KR100507545B1 (en) | 2002-09-03 | 2005-08-09 | 주식회사 엘지생명과학 | Hyaluronic acid derivatives and processes for preparing them |
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TW200524959A (en) * | 2003-09-08 | 2005-08-01 | Chugai Pharmaceutical Co Ltd | Hyaluronic acid modification product and drug carrier therefrom |
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AU2007280846A1 (en) | 2008-02-07 |
US8202986B2 (en) | 2012-06-19 |
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US20090312283A1 (en) | 2009-12-17 |
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